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Characterization of carotenoprotein from different shrimp shell waste for possible use as supplementary nutritive feed ingredient in animal diets
Journal Pre-proof Characterization of carotenoprotein from different shrimp shell waste for possible use as supplementary nutritive feed ingredient in animal diets Sandeep Shankar Pattanaik, Paramita Banerjee Sawant, K.A. Martin Xavier, Kiran Dube, Prem Prakash Srivastava, Vignaesh Dhanabalan, N.K. Chadha PII:
S0044-8486(19)30888-9
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734594
Reference:
AQUA 734594
To appear in:
Aquaculture
Received Date: 19 April 2019 Revised Date:
10 October 2019
Accepted Date: 10 October 2019
Please cite this article as: Pattanaik, S.S., Sawant, P.B., Xavier, K.A.M., Dube, K., Srivastava, P.P., Dhanabalan, V., Chadha, N.K., Characterization of carotenoprotein from different shrimp shell waste for possible use as supplementary nutritive feed ingredient in animal diets, Aquaculture (2019), doi: https:// doi.org/10.1016/j.aquaculture.2019.734594. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Characterization of carotenoprotein from different shrimp shell waste for
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possible use as supplementary nutritive feed ingredient in animal diets
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Sandeep Shankar Pattanaik, Paramita Banerjee Sawant*, K. A. Martin Xavier, Kiran Dube,
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Prem Prakash Srivastava, Vignaesh Dhanabalan and N. K. Chadha
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ICAR- Central Institute of Fisheries Education, Versova, Mumbai – 400061, Maharashtra,
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India
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*Corresponding author email: [email protected] Telephone: +919820731336
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Running title: Characteristics of carotenoprotein extracted from different shrimp shell waste
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Abstract
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Carotenoproteins from four different shrimp shell wastes Penaeus monodon,
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Parapenaeopsis stylifera, Metapenaeus affinis and Nematopalemon tenuipes were extracted
29
with the aid of papain enzyme and characterized by their protein, amino acid and carotenoid
30
content of the shell wastes and the antioxidant activities like DPPH, FRAP, ABTS radical
31
scavenging activity and reducing power assay of the carotenoprotein. Higher protein content of
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9.8 g 100g-1 and 9.2g 100g-1 was recovered from shell waste of Penaeus monodon and
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Parapenaeopsis stylifera respectively along with highest carotenoid content of 114 ± 0.02 µg g-
34
1
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Metapenaeus affinis. Highest antioxidant activity was found in the carotenoprotein extracted
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from the shell waste of P. stylifera which suggest that the antioxidant activity of carotenoids
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followed a concentration dependent pattern. The amino acid profile showed that
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carotenoprotein is a rich source of essential amino acids such as glutamic acid, aspartic acid,
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lysine and leucine. Among shell wastes, P.stylifera shell waste was calculated to be superior as
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it contained higher amount of essential amino acids and exhibited higher antioxidant activity in
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terms of protein, carotenoid as well as radical scavenging and reducing power and it could
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serve as a supplementary nutritive feed ingredient in animal diets. This would help in
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utilization of crustacean (shrimp) shell waste for formulating low cost feed for ornamental fish
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and also encourage shrimp processing industries to utilize of the same in order to control
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pollution of land and water.
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Keywords: shrimp shell waste; carotenoproteins; astaxanthin; essential amino acids;
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antioxidant activity; enzymatic hydrolysis; animal diet
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1. Introduction
in Parapenaeopsis stylifera followed by 100.6±0.02 µg g-1 from the shell waste of
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Carotenoproteins are stable products in which unstable carotenoids bind to the
50
hydrophobic sites of the protein that make the carotenoids more stable (Ghidalia, 1985).
51
Crustaceans are major sources of carotenoprotein which is mainly found in their ovaries and
52
eggs as carotenolipoproteins and in their exoskeletons as chitinocarotenoids and crustacyanins.
53
Crustacyanin extracted from shell waste of lobster is composed of two different stalks of
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astaxanthin bound together with proteins (Gamiz-Hernandez et al., 2015, Supplementary
55
figure-1). Waste from the shrimp industry as well as other crustacean industry is an excellent
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source of carotenoprotein and should be appropriately utilized as these are highly perishable
57
and create environmental pollution if dumped into water bodies. According to Yan and Chen
58
(2015), 6-8 million tons of shell waste is generated per year globally, from which
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approximately 1.5 million tons of shell wastes are generated from Asia alone. Moreover, these
60
are storehouse of many bioactive compounds like carotenoids, antioxidants, minerals, enzymes,
61
chitin, etc. (Coward-Kelly et al., 2006; Diaz-Rojas et al., 2006; Sachindra et al., 2006, 2007).
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These are capable of enhancing the growth and the immunity of cultured species (Weeratunge
63
and Perera, 2016). Hence, these discards can be reused in shell biorefineries to augment income
64
of income for shrimp farmers along with serving a dual purpose of decreasing the cost of feed
65
in aquaculture (Yan and Chen, 2015).
66
Moreover, carotenoprotein is composed of many essential amino acids such as
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glutamic acid, aspartic acid, lysine, leucine (Simpson and Haard 1985; Armenta & Guerrero-
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Legarreta, 2009). Since carotenoids (mainly astaxanthin) act as an antioxidant by preventing
69
cells from oxidative damage (Bendich and Olson, 1989; Tacon, 1981), they are instrumental in
70
protection against cardio-vascular disease and age-related phenomena caused by oxidative
71
damages (Haliwell, 1996).
72
Many methods have been standardized to extract the carotenoprotein from
73
shrimp shell waste. Due to the fat-soluble property of carotenoids, extraction of carotenoid
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using different vegetable oils like sunflower oil, groundnut oil, gingerly oil, mustard oil,
75
soybean oil, coconut oil, rice bran oil, palm oil as well as cod liver oil, krill oil, etc. have also
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been attempted (Chen and Meyers, 1982; Shahidi and Synowiecki, 1991; Sachindra and
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Mahendrakar,2005; Handayani et al., 2008). Since carotenoprotein extract is found to be more
78
stable than carotenoid (Cano-Lopez et al., 1987), enzymatic hydrolysis of shell waste could be
79
a possible method of extracting carotenoid pigments along with protein and the resulting
80
sample can be used as a dietary protein as well as a pigment source in aquafeed industry.
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During processing of chitin, trials have been made to extract protein from the shell using
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proteolytic enzymes as well as by bacterial degradation of protein (Shimahara et al., 1982;
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Simpson et al., 1994). Many researchers have used different proteolytic enzymes from
84
commercially available sources i:e, trypsin, pepsin, papain (Chakrabarty, 2002), trypsin from
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Albacore tuna spleen (Poonsin et al., 2017), commercial enzymes from different Aspergillus
86
species (Lee et al., 1999), Atlantic cod trypsin or bovine trypsin (Cano-Lopez et al., 1987),
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protease from hepatopancreas (Senphan et al., 2014) to break the protein-pigment bond to
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increase the carotenoid concentration in the extraction process or to split the chitin-pigment
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interaction to obtain more protein enriched pigment concentration (Hansen and Llanes, 1994).
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In india, waste produced through shrimp processing is one of the largest
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industrial wastes causing environmental pollution. These wastes can also act as a substrate for
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microbial growth (Sindhu and Sherief, 2011). On the other hand, these are a rich source of
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nutrients which can be used as animal feed suppliements either as bait or as fertilizer, as well as
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in chitin production (Yan and Chen, 2015). Hence, the present study has been conducted with
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the aim of evaluating the nutritional profile of carotenoprotein extracted from shell waste of
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four different shrimp species (i:e, Penaeus monodon, Metapenaeus affinis, Parapenaeopsis
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stylifera, Nematopalaemon tenuipes) using papain through enzymatic hydrolysis to
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characterize the resultant carotenoproteins. Furthermore, the study reveals the ability of shrimp
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shell waste to be used as a nutraceutical in the fish feed.
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2. Materials and methods:
102
2.1. Materials:
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Fresh shell waste of four shrimp species Penaeus monodon, Parapenaeopsis stylifera,
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Metapenaeus affinis, Nematopalemon tenuipes was collected from Versova landing center,
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Mumbai, India and transported in iced condition to the fish processing laboratory of ICAR-
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Central Institute of Fisheries Education, Mumbai. Fresh shell waste (cephalothorax and
107
carapace) were used for the extraction of carotenoprotein. Papain enzyme (a cysteine protease
108
of the peptidase C1 family, Molecular wt. 23406 Da) and all other reagents used for extraction
109
of carotenoprotein and its characterization were procured from Hi-media, E-Merck and
110
Qualigens. India.
111
2.2. Extraction of carotenoprotein:
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Shrimp shell waste was washed with chilled water (4 ± 1 °C), decanted and drained
113
with muslin cloth before extraction of carotenoprotein. Washed shell waste was then pulverized
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finely in a blender and weighed. Homogenate was prepared by adding water in grounded shell
115
waste @ 1:2 ratio (w/v) and this was further homogenized @ 9000 rpm for 2 min in a
116
homogenizer. pH of the homogenate was adjusted to 6.5 using 0.1N HCl. It was then
117
hydrolyzed using papain enzyme with an E/S ratio of 1:100 at 50 °C for 1 hr. The mixture was
118
heated for the termination of the hydrolysis reaction in water bath at 95 °C for 15 minutes
119
followed by filtration through Whatman filter paper no. 41. This mixture is referred to as
120
carotenoprotein. These carotenoprotein samples extracted from different shrimp shell wastes
121
were analyzed for protein, amino acid, carotenoid and antioxidant assay.
122
2.3. Protein content in shell waste:
123
Protein content in carotenoprotein was estimated using Biuret method
124
(Robinson & Hodgen, 1940) and expressed as g 100 g-1.
125
2.4. Carotenoid content in shell waste:
126
Total carotenoid present in shell waste was estimated according to the method
127
of Simpson and Haard (1985). 25 g of the sample was taken and carotenoid was repeatedly
128
extracted using acetone till the sample became colorless. The acetone extracts were pooled and
129
40 ml of petroleum ether (BP 40-60 ˚C) was added to it for phase separation. Petroleum ether
130
solution was separated out using separating funnel and repeatedly washed with 0.1 % NaCl
131
solution to remove the traces of acetone and filtered through anhydrous sodium sulphate to
132
remove traces of moisture. It was then vacuum dried at 40 ˚C, petroleum ether was added up to
133
a known volume and the absorbance was measured at 468 nm using an UV- VIS
134
spectrophotometer (Analytical Technologies). The carotenoid concentration (astaxanthin) was
135
calculated as
136
Carotenoid content (µg astaxanthin g-1 sample) =
137
×
. ×
×
Where A is the absorbance at 468nm, 0.2 is the absorbance value of the 1µg ml-1
138
astaxanthin standard.
139
2.5. Amino acid analysis:
140
Total amino acid composition was determined using a high-resolution Q-TOF
141
mass spectrometer equipped with an ion exchange column, G4220B quaternary pump, a 20 µl
142
injection valve and an HPLC-Diode Array Detector (DAD). Mobile phase A contained a
143
gradient elution with amino acid buffer and B had organic solvent (MeOH +ACN + H2O). The
144
flow rate was constant at 1.5 ml min-1, and the column temperature was set at 40 °C. Samples
145
were taken for hydrolysis in 6 N HCl in evacuated sealed test tubes at 110 °C for 24 h. After 1
146
min isocratic step at 2 % B, elution was started with a linear gradient of B from 2% to 57% in
147
13.40 min, this % of B was maintained for 0.1 min, then B was linearly increased to 100%
148
from 57 % in 0.1 min, held at 100% of B from 13.50 to 15.70 min, and finally the B content
149
was lowered to 2% and total cycle time of 18 min was set. The software used for MS data
150
analysis was Mass Hunter Qualitative Analysis B.06 (Agilent Technologies, Santa Clara, CA,
151
USA). Presence of amino acids was confirmed by comparing the fragmentation patterns and
152
retention times of samples with those of authentic standards. The identification and
153
quantification of amino acids was performed in comparing the peak areas of the corresponding
154
mass traces with those of authentic standards. The results were expressed in terms of mg amino
155
acid g-1 of shell waste.
156
2.6. Antioxidant activities of carotenoproteins from different shrimp shell wastes
157
2.6.1. 2,2-Diphenyl 1-picrylhydrazyl (DPPH) radical scavenging activity
158
DPPH radical scavenging activity of shrimp shell extracts was performed
159
according to the method of Brand-Williams et al. (1995) with some modifications. 24 mg
160
DPPH was mixed with 100 ml methanol as the stock solution and then stored at -20 °C until
161
used for further analysis. 10 ml stock solution was mixed with 45ml methanol to prepare the
162
working solution. 150 µl of carotenoprotein from shell waste was taken and mixed with 2850µl
163
of DPPH solution and kept in the dark for 30 min. The absorbance of the reaction mixture was
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taken at 517 nm. × 100
165
DPPH radical scavenging activity =
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Where, A0= Absorbance of control; A1= Absorbance of sample
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2.6.2. Ferric Reducing Antioxidant Power Assay
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The FRAP assay was determined according to the procedure of Benzie and Strain
169
(1996) with some modifications. The FRAP reagent (25 ml of 0.3M acetate buffer, pH 3.6, 2.5
170
ml of 10 mM TPTZ in 40 mM HCl, and 2.5 ml of 20 mM FeCl3·6H2O) was prepared fresh
171
daily and was warmed at 37 °C in a water bath prior to use. 150 µl of sample was added to 2.85
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ml of the FRAP reagent. After 4 min, the absorbance of the reaction mixture was recorded at
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593 nm. The standard curve was constructed using iron (Fe2+) sulfate solution (100–2000 µM),
174
and results were expressed as µ mol Fe2+ g-1 wet weight of the sample. All the samples were
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taken in triplicate and the mean values were calculated.
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2.6.3. ABTS radical scavenging activity
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2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt radical
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scavenging activity of the carotenoprotein from different shell wastes was measured by the
179
method of Arnao, Cano, & Acosta (2001). The stock solutions included 7.4mM ABTS solution
180
and 2.6mM potassium persulfate solution. The two stock solutions in equal quantities were
181
mixed to prepare the working solution and then it was allowed to react for 12 h at room
182
temperature in dark. The solution was mixed with 1 ml ABTS solution and 60 ml methanol to
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get an absorbance of 1.1 ±0.02 units at 734nm using the UV-visible spectrophotometer
184
(µQUANT Biotek). Carotenoprotein from shell waste solution of 150 µl was allowed to react
185
with 2850 µl of the ABTS solution for 2h in a dark condition while the ABTS solution was
186
prepared for each assay freshly. The absorbance was measured at 734 nm using UV
187
spectrophotometer. The standard curve was linear between 200 and 1000µM Trolox. Results
188
were expressed in µM Trolox equivalents (TE) g-1 of sample used.
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2.6.4. Reducing power assay
190
The reducing power of carotenoprotein
was determined according to the
191
method of Wu et al., (2003) with slight modifications. 2ml of carotenoprotein at different
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concentrations (0.5%, 1%, 2%, 3%, 4% and 5%) were added to 2ml of 0.2M phosphate buffer
193
(pH 6.6) and 2ml of 1% potassium ferricyanide. The reaction mixture was incubated at 50°C
194
for 20 min. Then 2ml of 10% TCA was added to the mixture, followed by centrifugation at
195
3000 rpm (Eltek centrifuge MP 400R, Electrocraft, India). A volume of 2ml (from each
196
incubated mixture) was mixed with 2ml of distilled water and 0.4ml of 0.1% ferric chloride in
197
a test tube. The mixture was allowed to react for 10 min and the absorbance of the solution was
198
taken at 700 nm using UV-visible spectrophotometer (µQUANT Biotek). Increasing
199
absorbance of the reaction mixture indicated the increasing reducing power.
200
201
2.7. Determination of phenolic and other polar compounds by HPLC-DADESI-QTOF-MS
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The extracts were reconstituted before analysis in ethanol-water (1:1, v/v) at a
203
concentration of 10,000 mg l-1 and filtered using regenerated cellulose syringe-filters of 0.2µm
204
pore size (Millipore, Bedford, MA, USA). Carotenoprotein extracted from the four different
205
shrimp shell wastes were analysed using High-Performance Liquid Chromatography coupled to
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electrospray ionization quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).
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An Agilent G6550A series Rapid Resolution Liquid Chromatographer coupled to a diode-array
208
detector (DAD) was used for the chromatographic determination. C18 column (4.6 × 150 mm,
209
1.8µm) (Model G1316C) was used from chromatographic separation at a flow rate of 0.3ml
210
min-1 using an injection volume of 3µl. The mobile phases were acidified water (0.1% formic
211
acid, v/v) and acetonitrile as solvent A and B, respectively. The following multi-step linear
212
gradient was used in order to achieve efficient separation: 0.0 min [A: B 95/5], 20.0 min [A: B
213
5/95], 25.0 min [A: B 5/95], 26.0 min [A: B 95/5], and 30 min [A: B 95/5]. Finally, initial
214
conditions were kept for 4 min at the end of each analysis to equilibrate the system before the
215
subsequent injection. The column temperature and auto-sampler compartment were set at 45
216
°C and 4 °C, respectively. Detection was performed in negative ionization mode over a mass
217
range from 125 to 1200 m/z. Ultrahigh pure nitrogen was used as drying and nebulizing gas.
218
The operating parameters for the separation were: drying gas temperature, 250 °C; drying gas
219
flow rate, 13 l min-1; nebulizer pressure, 241.31 kPa; nebulizer gas temperature, 250 °C;
220
nebulizer gas flow, 11 l min-1; capillary, 2500 V; fragmentor, 175 V; nozzle voltage, 1000 V;
221
skimmer, 65 V and octopole radiofrequency voltages, 750 V. All operations were processed
222
through Mass Hunter Qualitative Analysis B.06.00 (Agilent Technologies, Palo Alto, CA,
223
USA).
224 225
226
2.7. Statistical analysis
227
Experiments were run in triplicate using four different shell waste samples and all data
228
were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test to
229
compare the means. Statistical analysis was performed using the Statistical Package for Social
230
Science (SPPS 16.0 for windows).
231
3. Results and Discussion
232
Shrimp shells are major source of protein, carotenoid, calcium, chitin, amino acids as well as
233
antioxidants (Yan and Chen, 2015; Weeratunge and Perera, 2016; Sachindra et al., 2006,
234
2007). These can be used as nutraceuticals in fish feed as animal sources act efficiently as
235
nutraceuticals next to externally used antibiotics (Radinnurafiqah et al., 2016). Paital, (2016)
236
also reported fish and other aquatic organisms as an efficient cost-effective nutraceutical which
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can be recommended in clinical cases to replace the externally used antibiotics. The present
238
study helps in screening the effective shell waste that can be used for the above said purposes.
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3.1. Protein content in shrimp shell waste
240
Protein content in shrimp shell waste from four different species, are presented in Table-1,
241
have been found in the range of 5.6-9.8% (on wet weight basis). Protein content was highest in
242
shell waste of P.monodon (9.8 g 100 g-1) followed by P.stylifera (9.2 g100 g-1) and lowest in
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M.affinis (5.6g 100 g-1). Chakrabarti (2002) reported protein content of brown shrimp
244
(Metapenaues monocerous) shell waste as 8-10g 100g-1 and that in shell waste of Metapenaeus
245
endeavor was found to be 14.2 -15.2 g 100g-1 (Ariyani and Buckle, 1991). Similarly, protein
246
content in the by-products of northern pink shrimp (Pandalus borealis) was 9.3g 100g-1 and
247
that of spotted shrimp (Trachypene curvirostris) was 11.6 g 100 g-1 (Heu et al., 2003). Babu et
248
al. (2008) examined carotenoprotein contents of head waste from Penaeus monodon, M.
249
monocerus and P. indicus as 11.2 g 100 g-1, 11.3 g 100 g-1 and 12.3 g 100g-1 protein content
250
respectively.
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3.2 Carotenoid content of proteins extracted from different shell waste
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Carotenoid content in the shell waste from different species of shrimp varied significantly
253
(p<0.05) which is shown in Table-1. The highest carotenoid content was recorded from
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P.stylifera shell waste (114 ± 0.02 µg g-1) while the lowest was recorded in N.tennupes (40.3 ±
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0.01 µg g-1). In M.affinis, it was 100.6 ± 0.02 µg g-1. The carotenoid content of P.monodon
256
shell waste was 51 ± 0.02 µg g-1. Carotenoid content in crustaceans vary according to species
257
(Lambertsen et al., 1971). The carotenoid content in tiger prawns (P. monodon) from waters of
258
the Indo-Pacific region varied from 23 to 331 µg g-1 in the exoskeleton. Sachindra et al. (2005)
259
reported the carotenoid concentration in the carapace of P. monodon, P. indicus, Metapenaeus
260
dobsonii and Parapenaeopsis stylifera as 86.6, 59.8, 83.3, 104.7 µg g-1 respectively. Among
261
these, carotenoid concentration was found to be highest in the meat, head as well as carapace of
262
P. stylifera. Similar results have been obtained in the present study wherein, the carotenoid
263
content of four different shrimp shell wastes from P. monodon, M. affinis, P. stylifera and N.
264
tenuipes were 51, 100.6, 114, 40.3 µg g-1 respectively. Highest carotenoid yield was obtained
265
from the P. stylifera shell waste.
266
3.3. Amino acids profile of the carotenoprotein
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Amino acid composition of carotenoprotein extracted from four different shell wastes are
268
represented in Table-2. Total amino acid content was highest in the carotenoprotein of P.
269
stylifera (478.30 mg g-1) and lowest was found in M.affinis (169.24 mg g-1). Amount of total
270
essential amino acid was found to be highest in P. stylifera (198.38 mg g-1). Among all amino
271
acids, glutamic acid is mainly found in higher quantity in all the carotenoproteins. Glutamic
272
acid, hydroxyproline, leucine, isoleucine, lysine, valine mainly contributes more than 50% of
273
amino acid content. Glutamine is found only in P. stylifera shell waste at a concentration of
274
1.10 mg g-1 shell waste. According to Armenta and Guerrero-Legarreta (2009),
275
carotenoproteins extracted from fermented and non-fermented Pacific white shrimp waste were
276
rich in aspartic acid and glutamic acids as well as in leucine and lysine. Carotenoprotein
277
recovered with and without the aid of bluefish trypsin contained high amount of glutamic acid/
278
glutamine (13.00 and 13.25%) and aspartic acid/ asparagines (11.18 and10.43%) (Klomklao et
279
al, 2009). Simpson and Haard (1985) also found that glutamic acid and aspartic acid were the
280
dominant amino acids in carotenoproteins isolated from shrimp wastes with and without the aid
281
of bovine trypsin. The present study reported higher carotenoprotein in SSW of P.stylifera
282
(478.3 mg g-1 shell waste) out of which, 198.38 mg g-1 was contributed by essential amino
283
acids. On the contrary, lowest amino acid content was recorded in carotenoprotein from M.
284
affinis shell waste in the present study. Similarly, Senphan et al. (2014) also reported highest
285
contribution of glutamic acid, aspartic acid, alanine, leucine, lysine, isoleucine, valine and
286
lowest content of cysteine and hydroxyproline in Pacific white shrimp (L. vannamei).
287
3.3. Antioxidant activity of different shell waste
288
DPPH ((2, 2-Diphenyl 1-picryl hydrazyl) radical scavenging activity of carotenoprotein from
289
different shell wastes are shown in Table-1. DPPH radical scavenging activity has been widely
290
used for the determination of primary antioxidant activity (Limsuwanmanee et al., 2014). The
291
present study reported highest activity in carotenoprotein extracted from P. stylifera (72.96%)
292
and the lowest in N. tenuipes (16.06%). In spite of that, carotenoids extracted from different
293
shell waste showed lower radical scavenging activity than BHA (91.4±0.008%), which is
294
normally considered as a standard for DPPH radical scavenging activity. Sila et al. (2014)
295
reported that carotenoprotein extracted from shrimp P. longirostris by-products showed higher
296
DPPH radical scavenging activity. Further, they concluded that the antioxidant activity of
297
carotenoprotein is mainly dependant on carotenoid content. Therefore, higher carotenoid
298
content in P. stylifera SSW than the other shell wastes can be correlated with better DPPH
299
radical scavenging activity in the former.
300
Ferric Reducing Antioxidant Power Assay of carotenoprotein from various shell wastes
301
is shown in Table-1 and expressed as µM Fe2+ g-1. Significant differences (P < 0.05) were
302
evident between the carotenoprotein content of different shell wastes and FRAP activity was
303
found to be highest in P. stylifera (0.979 ± 0.01 µM Fe2+ g-1), followed by M. affinis shell waste
304
(0.95 ± 0.004 µM Fe2+ g-1) while the lowest activity was obtained in P. monodon shell waste
305
(0.635 ± 0.001 µM Fe2+ g-1). Sowmya and Sachindra (2012) estimated antioxidant activity of
306
shrimp processing discards and suggested that carotenoids have a major role in the scavenging
307
of free radicals. Similar trends have also been reported in FRAP activity in the present
308
experiment, wherein highest and lowest FRAP activity was recorded in the waste from P.
309
stylifera (0.979 µM Fe2+ g-1) and N. tenuipes, respectively.
310
The ABTS radical scavenging activity assay can be applied to both lipophilic and
311
hydrophilic compounds and has been widely used as an antioxidant activity assay (Re et al.,
312
1999). 2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt radical
313
scavenging activity of carotenoprotein from different shrimp shell wastes is shown in Table-1.
314
ABTS activity was found to be significantly different in all samples and varied from 163.25 ±
315
2.02 mM Trolox g-1 to 396 ± 3.92 mM Trolox g-1 among the shell wastes. It was highest in P.
316
stylifera shell waste (396.583 ± 3.92 mM Trolox g-1) and the lowest value was recorded in N.
317
tenuipes shell waste (163.25 ± 2.02 mM Trolox g-1). Higher ABTS activity in P. stylifera can
318
be explained by the occurrence of higher amount of carotenoid content in it, which helped in
319
scavenging the metal ions (Sowmya and Sachindra, 2012).
320
The above results suggest that antioxidative activities of carotenoids in carotenoproteins
321
of different shell wastes followed a concentration dependent pattern wherein P. stylifera (with
322
higher carotenoid concentration) showed higher antioxidant activity compared to all others;
323
lowest being in N. tenuipes. Carotenoid (mainly astaxanthin), has the ability to quench, trap as
324
well as scavenge free radicals and it also neutralizes free radicals by adding them into its own
325
double bond (Higuera-Ciapara, Felix-Valenzuela and Goycoolea, 2006). Thus, carotenoprotein
326
had higher radical scavenging activity as well as chelating capability when the carotenoid
327
content was higher in them, in accordance to results obtained by Sowmya and Sachindra (2012)
328
and Senphan et al. (2014). Moreover, carotenoprotein contains both carotenoid as well as
329
protein where the carotenoids and peptides in the protein act as an antioxidant. Cephalothorax
330
extracts are found to be responsible for the radical scavenging properties due to presence of
331
peptides in them (Binsan et al., 2008). Similarly, peptides from protein hydrolysates obtained
332
from shrimp shell wastes played a significant role as antioxidants (He et al., 2006).
333
Reducing power assay is mainly used to evaluate the ability of antioxidant to
334
transfer electron or hydrogen (Yildirim et al., 2003). Again, reducing power assay has
335
been found to be higher in the carotenoprotein from P. stylifera shell waste and lowest
336
in that of N. tenuipes shell waste, wherein, the OD value increased from 1.0025 to
337
3.0445, while N. tenuipes exhibited lesser absorbance from 0.866 to 1.1245 which has
338
been shown in Figure 1. Reducing power is mainly affected by bioactive compounds
339
present in shrimp shell waste, such as carotenoids, phenolic compounds,
340
chitooligosaccharides etc, which can donate electrons easily and terminate the radical
341
chain reactions (Ghorbel-Bellaaj et al., 2012). Therefore, bioactive compounds present
342
in the carotenoid and protein content of shell waste help in reducing or scavenging free
343
radicals into more stable forms thereby acting as an antioxidant. Seymour et al. (1996)
344
also reported the presence of natural antioxidants like phenolic compounds in different
345
shrimp wastes.
346
3.4 Characterization of carotenoid profile by HPLC-DAD-ESI-QTOF MS analysis
347
The carotenoprotein extracted from Parapeneopsis stylifera was evaluated for the
348
identification of carotenoid compounds by HPLC-DAD-ESI-QTOF MS analysis and the
349
HPLC chromatogram profile was shown in Figure 2. A total of 5 carotenoid derivatives,
350
including all keto carotenoid, xanthophyll and hydroxylated carotenoid were identified. Peak
351
identification was carried out based on retention time behavior and absorption spectra which
352
were shown in Table-3. All peaks in the chromatogram were detected in the retention time from
353
19.716 to 21.229 min. Peak 1 with a molecular ion at m/z 597.3905 and four fragments at m/z
354
597.3905, 598.3943, 599.398, 600.4022 corresponding to MH+ ions respectively was identified
355
as astaxanthin. Lin et al. (2005) also reported the presence of astaxanthin in spear shrimp shells
356
(Parapenaeopsis hardwickii). Three compounds (Beta-carotene, cryptoxanthin and Lutein) at
357
m/z 537.4465 and 553.4374 were categorized as xanthophyll, a derivative of carotenoid.
358
MS/MS spectra yielded three fragments corresponding to (M+H)+ and (M+Na)+ ions detected
359
in Peak 2, 4 and 5. Breithaupt (2004) also characterized the shrimp (Pandalus borealis) and
360
revealed the incidence of xanthophyll in shrimp extract. Peak 3 has retention time of 21.112
361
min, with a molecular ion at m/z 537.4465 and MS/MS spectra provided three fragments
362
resulting (M+H) + ions described as Lycopene.
363
Hence the properties of carotenoproteins described in the present work can
364
maximize the utilization of shrimp shell to cope up with the increased shell waste generation
365
which can reduce the heavy load of disposal into the ocean or landfill (Yan and Chen, 2015).
366
4. Conclusion
367
Carotenoprotein, a stable form of carotenoid, can be efficiently extracted through enzymatic
368
hydrolysis using papain enzyme, can serve as an effective antioxidant as well as being a rich
369
source of essential amino acid and carotenoid. These extracted protein-carotenoid complexes
370
can be used as nutraceuticals in feed to produce cost effective and antioxidant rich diets for
371
enhancing immunity and survival of the cultured species. Superiority of the carotenoprotein
372
extracted from shell waste of parapeneopsis stylifera has been proven in the present study in
373
terms of higher quantity of essential amino acids, carotenoids and protein content and enhanced
374
antioxidant activities as compared to the carotenoprotein extracted from shell waste of Penaeus
375
monodon, Metapenaeus affinis, Nematopalemon tenuipes. Further, research may be conducted
376
on the concept of using carotenoprotein extracted from parapeneopsis stylifera as a feed
377
ingredient, being a cost-effective nutraceutical and colour enhancer (both act as an antioxidant
378
as well as a source of carotenoids and essential amino acid).
379
Acknowledgements
380
The authors are thankful to the Director and Vice-chancellor, ICAR-Central Institute of
381
Fisheries Education, Mumbai, India for the necessary support and encouragement.
382
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383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414
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Table 1. Characterization of carotenoprotein extracted from shell waste of different shrimp species (on wet weight basis) Shell waste of different shrimp species Parameter P.monodon
M.affinis
P.stylifera
N.tenuipes
Protein content (g 100 g-1)
9.80 ± 0.02d
5.60 ± 0.01a
9.20 ± 0.03c
8.90 ± 0.02b
carotenoid content (µg/ g sample)
51 ± 0.02b
114 ± 0.03d
40.30 ± 0.01a
DPPH Activity (%) FRAP activity (µM Fe2+/g) ABTS Activity (mM Trolox/g)
100.6 ± 0.02c
25.13 ± 0.01b
27.47 ± 0.01b
72.96 ± 0.04c
16.06 ±0.02a
0.63± 0.01a
0.95 ± 0.04c
0.97 ± 0.01c
0.82 ± 0.01b
294.08 ± 1.39c
200.77 ± 1.46b
396.58 ± 3.92d
163.25 ± 2.02a
Values are expressed in terms of Mean ± SE a,b,c,d values in a column with different superscripts differ significantly (p < 0.05)
Table 2. Amino acid composition of carotenoprotein from different shell wastes
Amino acid
Pm
Ps
Ma
Na
Arginine*
10.54
12.97
7.05
9.46
Threonine*
11.07
19.81
4.31
11.53
Valine*
19.14
31.41
23.79
22.08
Isoleucine *
14.99
25.70
0.00
14.85
Leucine*
24.10
39.20
26.24
24.59
Lysine*
26.70
39.72
8.18
26.60
Methionine*
4.54
8.85
8.75
4.65
Phenaylalanine*
12.31
20.72
6.37
10.62
Alanine
26.82
41.46
9.05
26.09
Aspartic acid
32.38
54.42
9.53
33.17
Glutamine
0.00
1.10
0.00
0.00
Glutamic acid
57.20
87.85
5.16
54.19
Glycine
21.23
35.68
4.61
18.71
Hydroxyproline
31.82
46.77
5.81
32.90
Serine
(mg/g shell waste)
10.06
12.64
8.10
7.86
A
123.40
198.38
84.69
124.37
TNEEB
179.51
279.93
42.25
172.92
Total Amino Acid
302.90
478.30
126.93
297.28
TEAA
*Essential amino acid in adults A
Total Essential Amino Acid
B
Total Non-Essential Amino Acid
Pm = Penaeus monodon; Ma = Metapenaeus affinis; Ps = Parapeneopsis stylifera; Nt = Nematopalemon tenuipes
Table 3. Characterization of carotenoid profile by HPLC-DAD-ESI-QTOF MS analysis
Peaks
RT (Retention Time)
Molecular formula
Structure
Mass spectrometry (m/z)
MS/MS fragments
Compound label
Major types (representative components) of carotenoid compounds
Astaxanthin
Keto carotenoid
1
19.716
C40H52O4
597.3905
597.3905 598.3943 599.398 600.4022
2
21.112
C40H56
537.4465
537.4465 538.4477 539.4437
Beta-carotene
Hydroxylated carotenoid (xanthophyll)
Lycopene
Carotenoid
3
21.112
C40H56
537.4465
537.4465 538.4477 539.4437
4
21.396
C40H56O
553.4374
553.4374 554.4483 575.4077
Cryptoxanthin
Hydroxylated carotenoid (xanthophyll)
569.4349
569.4349 570.4378 591.4085 592.4228
Lutein
Xanthophyll
5
21.229
C40H56O2
3.5
3
OD value
2.5
2
P.monodon M.affinis
1.5
P.stylifera N.tenuipes
1 0.5
0 0.5
1
2
3
4
5
concentration of carotenoprotein
Fig.1. Reducing power activity of carotenoprotein of the shell of different shrimp species *Values in bars with different superscripts differ significantly (P < 0.05)
Fig.2. Carotenoid profile of the lipid extract from P. Stylifera shrimp waste as determined by HPLC-DAD
Highlights •
Four different shrimp species shell waste were comparatively evaluated.
•
Carotenoprotein was extracted through enzymatic hydrolysis using papain enzyme.
•
Amino acid, carotenoid, carotenoid profile and antioxidant properties of four different shrimp shell waste were studied for the first time.
•
Superiority of carotenoprotein extracted from Parapeneopsis stylifera has been found.
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DOI:
https://doi.org/10.1016/j.aquaculture.2019.734594
Reference:
AQUA 734594
To appear in:
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Received Date: 19 April 2019 Revised Date:
10 October 2019
Accepted Date: 10 October 2019
Please cite this article as: Pattanaik, S.S., Sawant, P.B., Xavier, K.A.M., Dube, K., Srivastava, P.P., Dhanabalan, V., Chadha, N.K., Characterization of carotenoprotein from different shrimp shell waste for possible use as supplementary nutritive feed ingredient in animal diets, Aquaculture (2019), doi: https:// doi.org/10.1016/j.aquaculture.2019.734594. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Characterization of carotenoprotein from different shrimp shell waste for
2
possible use as supplementary nutritive feed ingredient in animal diets
3
Sandeep Shankar Pattanaik, Paramita Banerjee Sawant*, K. A. Martin Xavier, Kiran Dube,
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Prem Prakash Srivastava, Vignaesh Dhanabalan and N. K. Chadha
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ICAR- Central Institute of Fisheries Education, Versova, Mumbai – 400061, Maharashtra,
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India
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*Corresponding author email: [email protected] Telephone: +919820731336
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Running title: Characteristics of carotenoprotein extracted from different shrimp shell waste
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Abstract
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Carotenoproteins from four different shrimp shell wastes Penaeus monodon,
28
Parapenaeopsis stylifera, Metapenaeus affinis and Nematopalemon tenuipes were extracted
29
with the aid of papain enzyme and characterized by their protein, amino acid and carotenoid
30
content of the shell wastes and the antioxidant activities like DPPH, FRAP, ABTS radical
31
scavenging activity and reducing power assay of the carotenoprotein. Higher protein content of
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9.8 g 100g-1 and 9.2g 100g-1 was recovered from shell waste of Penaeus monodon and
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Parapenaeopsis stylifera respectively along with highest carotenoid content of 114 ± 0.02 µg g-
34
1
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Metapenaeus affinis. Highest antioxidant activity was found in the carotenoprotein extracted
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from the shell waste of P. stylifera which suggest that the antioxidant activity of carotenoids
37
followed a concentration dependent pattern. The amino acid profile showed that
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carotenoprotein is a rich source of essential amino acids such as glutamic acid, aspartic acid,
39
lysine and leucine. Among shell wastes, P.stylifera shell waste was calculated to be superior as
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it contained higher amount of essential amino acids and exhibited higher antioxidant activity in
41
terms of protein, carotenoid as well as radical scavenging and reducing power and it could
42
serve as a supplementary nutritive feed ingredient in animal diets. This would help in
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utilization of crustacean (shrimp) shell waste for formulating low cost feed for ornamental fish
44
and also encourage shrimp processing industries to utilize of the same in order to control
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pollution of land and water.
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Keywords: shrimp shell waste; carotenoproteins; astaxanthin; essential amino acids;
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antioxidant activity; enzymatic hydrolysis; animal diet
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1. Introduction
in Parapenaeopsis stylifera followed by 100.6±0.02 µg g-1 from the shell waste of
49
Carotenoproteins are stable products in which unstable carotenoids bind to the
50
hydrophobic sites of the protein that make the carotenoids more stable (Ghidalia, 1985).
51
Crustaceans are major sources of carotenoprotein which is mainly found in their ovaries and
52
eggs as carotenolipoproteins and in their exoskeletons as chitinocarotenoids and crustacyanins.
53
Crustacyanin extracted from shell waste of lobster is composed of two different stalks of
54
astaxanthin bound together with proteins (Gamiz-Hernandez et al., 2015, Supplementary
55
figure-1). Waste from the shrimp industry as well as other crustacean industry is an excellent
56
source of carotenoprotein and should be appropriately utilized as these are highly perishable
57
and create environmental pollution if dumped into water bodies. According to Yan and Chen
58
(2015), 6-8 million tons of shell waste is generated per year globally, from which
59
approximately 1.5 million tons of shell wastes are generated from Asia alone. Moreover, these
60
are storehouse of many bioactive compounds like carotenoids, antioxidants, minerals, enzymes,
61
chitin, etc. (Coward-Kelly et al., 2006; Diaz-Rojas et al., 2006; Sachindra et al., 2006, 2007).
62
These are capable of enhancing the growth and the immunity of cultured species (Weeratunge
63
and Perera, 2016). Hence, these discards can be reused in shell biorefineries to augment income
64
of income for shrimp farmers along with serving a dual purpose of decreasing the cost of feed
65
in aquaculture (Yan and Chen, 2015).
66
Moreover, carotenoprotein is composed of many essential amino acids such as
67
glutamic acid, aspartic acid, lysine, leucine (Simpson and Haard 1985; Armenta & Guerrero-
68
Legarreta, 2009). Since carotenoids (mainly astaxanthin) act as an antioxidant by preventing
69
cells from oxidative damage (Bendich and Olson, 1989; Tacon, 1981), they are instrumental in
70
protection against cardio-vascular disease and age-related phenomena caused by oxidative
71
damages (Haliwell, 1996).
72
Many methods have been standardized to extract the carotenoprotein from
73
shrimp shell waste. Due to the fat-soluble property of carotenoids, extraction of carotenoid
74
using different vegetable oils like sunflower oil, groundnut oil, gingerly oil, mustard oil,
75
soybean oil, coconut oil, rice bran oil, palm oil as well as cod liver oil, krill oil, etc. have also
76
been attempted (Chen and Meyers, 1982; Shahidi and Synowiecki, 1991; Sachindra and
77
Mahendrakar,2005; Handayani et al., 2008). Since carotenoprotein extract is found to be more
78
stable than carotenoid (Cano-Lopez et al., 1987), enzymatic hydrolysis of shell waste could be
79
a possible method of extracting carotenoid pigments along with protein and the resulting
80
sample can be used as a dietary protein as well as a pigment source in aquafeed industry.
81
During processing of chitin, trials have been made to extract protein from the shell using
82
proteolytic enzymes as well as by bacterial degradation of protein (Shimahara et al., 1982;
83
Simpson et al., 1994). Many researchers have used different proteolytic enzymes from
84
commercially available sources i:e, trypsin, pepsin, papain (Chakrabarty, 2002), trypsin from
85
Albacore tuna spleen (Poonsin et al., 2017), commercial enzymes from different Aspergillus
86
species (Lee et al., 1999), Atlantic cod trypsin or bovine trypsin (Cano-Lopez et al., 1987),
87
protease from hepatopancreas (Senphan et al., 2014) to break the protein-pigment bond to
88
increase the carotenoid concentration in the extraction process or to split the chitin-pigment
89
interaction to obtain more protein enriched pigment concentration (Hansen and Llanes, 1994).
90
In india, waste produced through shrimp processing is one of the largest
91
industrial wastes causing environmental pollution. These wastes can also act as a substrate for
92
microbial growth (Sindhu and Sherief, 2011). On the other hand, these are a rich source of
93
nutrients which can be used as animal feed suppliements either as bait or as fertilizer, as well as
94
in chitin production (Yan and Chen, 2015). Hence, the present study has been conducted with
95
the aim of evaluating the nutritional profile of carotenoprotein extracted from shell waste of
96
four different shrimp species (i:e, Penaeus monodon, Metapenaeus affinis, Parapenaeopsis
97
stylifera, Nematopalaemon tenuipes) using papain through enzymatic hydrolysis to
98
characterize the resultant carotenoproteins. Furthermore, the study reveals the ability of shrimp
99
shell waste to be used as a nutraceutical in the fish feed.
100
101
2. Materials and methods:
102
2.1. Materials:
103
Fresh shell waste of four shrimp species Penaeus monodon, Parapenaeopsis stylifera,
104
Metapenaeus affinis, Nematopalemon tenuipes was collected from Versova landing center,
105
Mumbai, India and transported in iced condition to the fish processing laboratory of ICAR-
106
Central Institute of Fisheries Education, Mumbai. Fresh shell waste (cephalothorax and
107
carapace) were used for the extraction of carotenoprotein. Papain enzyme (a cysteine protease
108
of the peptidase C1 family, Molecular wt. 23406 Da) and all other reagents used for extraction
109
of carotenoprotein and its characterization were procured from Hi-media, E-Merck and
110
Qualigens. India.
111
2.2. Extraction of carotenoprotein:
112
Shrimp shell waste was washed with chilled water (4 ± 1 °C), decanted and drained
113
with muslin cloth before extraction of carotenoprotein. Washed shell waste was then pulverized
114
finely in a blender and weighed. Homogenate was prepared by adding water in grounded shell
115
waste @ 1:2 ratio (w/v) and this was further homogenized @ 9000 rpm for 2 min in a
116
homogenizer. pH of the homogenate was adjusted to 6.5 using 0.1N HCl. It was then
117
hydrolyzed using papain enzyme with an E/S ratio of 1:100 at 50 °C for 1 hr. The mixture was
118
heated for the termination of the hydrolysis reaction in water bath at 95 °C for 15 minutes
119
followed by filtration through Whatman filter paper no. 41. This mixture is referred to as
120
carotenoprotein. These carotenoprotein samples extracted from different shrimp shell wastes
121
were analyzed for protein, amino acid, carotenoid and antioxidant assay.
122
2.3. Protein content in shell waste:
123
Protein content in carotenoprotein was estimated using Biuret method
124
(Robinson & Hodgen, 1940) and expressed as g 100 g-1.
125
2.4. Carotenoid content in shell waste:
126
Total carotenoid present in shell waste was estimated according to the method
127
of Simpson and Haard (1985). 25 g of the sample was taken and carotenoid was repeatedly
128
extracted using acetone till the sample became colorless. The acetone extracts were pooled and
129
40 ml of petroleum ether (BP 40-60 ˚C) was added to it for phase separation. Petroleum ether
130
solution was separated out using separating funnel and repeatedly washed with 0.1 % NaCl
131
solution to remove the traces of acetone and filtered through anhydrous sodium sulphate to
132
remove traces of moisture. It was then vacuum dried at 40 ˚C, petroleum ether was added up to
133
a known volume and the absorbance was measured at 468 nm using an UV- VIS
134
spectrophotometer (Analytical Technologies). The carotenoid concentration (astaxanthin) was
135
calculated as
136
Carotenoid content (µg astaxanthin g-1 sample) =
137
×
. ×
×
Where A is the absorbance at 468nm, 0.2 is the absorbance value of the 1µg ml-1
138
astaxanthin standard.
139
2.5. Amino acid analysis:
140
Total amino acid composition was determined using a high-resolution Q-TOF
141
mass spectrometer equipped with an ion exchange column, G4220B quaternary pump, a 20 µl
142
injection valve and an HPLC-Diode Array Detector (DAD). Mobile phase A contained a
143
gradient elution with amino acid buffer and B had organic solvent (MeOH +ACN + H2O). The
144
flow rate was constant at 1.5 ml min-1, and the column temperature was set at 40 °C. Samples
145
were taken for hydrolysis in 6 N HCl in evacuated sealed test tubes at 110 °C for 24 h. After 1
146
min isocratic step at 2 % B, elution was started with a linear gradient of B from 2% to 57% in
147
13.40 min, this % of B was maintained for 0.1 min, then B was linearly increased to 100%
148
from 57 % in 0.1 min, held at 100% of B from 13.50 to 15.70 min, and finally the B content
149
was lowered to 2% and total cycle time of 18 min was set. The software used for MS data
150
analysis was Mass Hunter Qualitative Analysis B.06 (Agilent Technologies, Santa Clara, CA,
151
USA). Presence of amino acids was confirmed by comparing the fragmentation patterns and
152
retention times of samples with those of authentic standards. The identification and
153
quantification of amino acids was performed in comparing the peak areas of the corresponding
154
mass traces with those of authentic standards. The results were expressed in terms of mg amino
155
acid g-1 of shell waste.
156
2.6. Antioxidant activities of carotenoproteins from different shrimp shell wastes
157
2.6.1. 2,2-Diphenyl 1-picrylhydrazyl (DPPH) radical scavenging activity
158
DPPH radical scavenging activity of shrimp shell extracts was performed
159
according to the method of Brand-Williams et al. (1995) with some modifications. 24 mg
160
DPPH was mixed with 100 ml methanol as the stock solution and then stored at -20 °C until
161
used for further analysis. 10 ml stock solution was mixed with 45ml methanol to prepare the
162
working solution. 150 µl of carotenoprotein from shell waste was taken and mixed with 2850µl
163
of DPPH solution and kept in the dark for 30 min. The absorbance of the reaction mixture was
164
taken at 517 nm. × 100
165
DPPH radical scavenging activity =
166
Where, A0= Absorbance of control; A1= Absorbance of sample
167
2.6.2. Ferric Reducing Antioxidant Power Assay
168
The FRAP assay was determined according to the procedure of Benzie and Strain
169
(1996) with some modifications. The FRAP reagent (25 ml of 0.3M acetate buffer, pH 3.6, 2.5
170
ml of 10 mM TPTZ in 40 mM HCl, and 2.5 ml of 20 mM FeCl3·6H2O) was prepared fresh
171
daily and was warmed at 37 °C in a water bath prior to use. 150 µl of sample was added to 2.85
172
ml of the FRAP reagent. After 4 min, the absorbance of the reaction mixture was recorded at
173
593 nm. The standard curve was constructed using iron (Fe2+) sulfate solution (100–2000 µM),
174
and results were expressed as µ mol Fe2+ g-1 wet weight of the sample. All the samples were
175
taken in triplicate and the mean values were calculated.
176
2.6.3. ABTS radical scavenging activity
177
2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt radical
178
scavenging activity of the carotenoprotein from different shell wastes was measured by the
179
method of Arnao, Cano, & Acosta (2001). The stock solutions included 7.4mM ABTS solution
180
and 2.6mM potassium persulfate solution. The two stock solutions in equal quantities were
181
mixed to prepare the working solution and then it was allowed to react for 12 h at room
182
temperature in dark. The solution was mixed with 1 ml ABTS solution and 60 ml methanol to
183
get an absorbance of 1.1 ±0.02 units at 734nm using the UV-visible spectrophotometer
184
(µQUANT Biotek). Carotenoprotein from shell waste solution of 150 µl was allowed to react
185
with 2850 µl of the ABTS solution for 2h in a dark condition while the ABTS solution was
186
prepared for each assay freshly. The absorbance was measured at 734 nm using UV
187
spectrophotometer. The standard curve was linear between 200 and 1000µM Trolox. Results
188
were expressed in µM Trolox equivalents (TE) g-1 of sample used.
189
2.6.4. Reducing power assay
190
The reducing power of carotenoprotein
was determined according to the
191
method of Wu et al., (2003) with slight modifications. 2ml of carotenoprotein at different
192
concentrations (0.5%, 1%, 2%, 3%, 4% and 5%) were added to 2ml of 0.2M phosphate buffer
193
(pH 6.6) and 2ml of 1% potassium ferricyanide. The reaction mixture was incubated at 50°C
194
for 20 min. Then 2ml of 10% TCA was added to the mixture, followed by centrifugation at
195
3000 rpm (Eltek centrifuge MP 400R, Electrocraft, India). A volume of 2ml (from each
196
incubated mixture) was mixed with 2ml of distilled water and 0.4ml of 0.1% ferric chloride in
197
a test tube. The mixture was allowed to react for 10 min and the absorbance of the solution was
198
taken at 700 nm using UV-visible spectrophotometer (µQUANT Biotek). Increasing
199
absorbance of the reaction mixture indicated the increasing reducing power.
200
201
2.7. Determination of phenolic and other polar compounds by HPLC-DADESI-QTOF-MS
202
The extracts were reconstituted before analysis in ethanol-water (1:1, v/v) at a
203
concentration of 10,000 mg l-1 and filtered using regenerated cellulose syringe-filters of 0.2µm
204
pore size (Millipore, Bedford, MA, USA). Carotenoprotein extracted from the four different
205
shrimp shell wastes were analysed using High-Performance Liquid Chromatography coupled to
206
electrospray ionization quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).
207
An Agilent G6550A series Rapid Resolution Liquid Chromatographer coupled to a diode-array
208
detector (DAD) was used for the chromatographic determination. C18 column (4.6 × 150 mm,
209
1.8µm) (Model G1316C) was used from chromatographic separation at a flow rate of 0.3ml
210
min-1 using an injection volume of 3µl. The mobile phases were acidified water (0.1% formic
211
acid, v/v) and acetonitrile as solvent A and B, respectively. The following multi-step linear
212
gradient was used in order to achieve efficient separation: 0.0 min [A: B 95/5], 20.0 min [A: B
213
5/95], 25.0 min [A: B 5/95], 26.0 min [A: B 95/5], and 30 min [A: B 95/5]. Finally, initial
214
conditions were kept for 4 min at the end of each analysis to equilibrate the system before the
215
subsequent injection. The column temperature and auto-sampler compartment were set at 45
216
°C and 4 °C, respectively. Detection was performed in negative ionization mode over a mass
217
range from 125 to 1200 m/z. Ultrahigh pure nitrogen was used as drying and nebulizing gas.
218
The operating parameters for the separation were: drying gas temperature, 250 °C; drying gas
219
flow rate, 13 l min-1; nebulizer pressure, 241.31 kPa; nebulizer gas temperature, 250 °C;
220
nebulizer gas flow, 11 l min-1; capillary, 2500 V; fragmentor, 175 V; nozzle voltage, 1000 V;
221
skimmer, 65 V and octopole radiofrequency voltages, 750 V. All operations were processed
222
through Mass Hunter Qualitative Analysis B.06.00 (Agilent Technologies, Palo Alto, CA,
223
USA).
224 225
226
2.7. Statistical analysis
227
Experiments were run in triplicate using four different shell waste samples and all data
228
were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test to
229
compare the means. Statistical analysis was performed using the Statistical Package for Social
230
Science (SPPS 16.0 for windows).
231
3. Results and Discussion
232
Shrimp shells are major source of protein, carotenoid, calcium, chitin, amino acids as well as
233
antioxidants (Yan and Chen, 2015; Weeratunge and Perera, 2016; Sachindra et al., 2006,
234
2007). These can be used as nutraceuticals in fish feed as animal sources act efficiently as
235
nutraceuticals next to externally used antibiotics (Radinnurafiqah et al., 2016). Paital, (2016)
236
also reported fish and other aquatic organisms as an efficient cost-effective nutraceutical which
237
can be recommended in clinical cases to replace the externally used antibiotics. The present
238
study helps in screening the effective shell waste that can be used for the above said purposes.
239
3.1. Protein content in shrimp shell waste
240
Protein content in shrimp shell waste from four different species, are presented in Table-1,
241
have been found in the range of 5.6-9.8% (on wet weight basis). Protein content was highest in
242
shell waste of P.monodon (9.8 g 100 g-1) followed by P.stylifera (9.2 g100 g-1) and lowest in
243
M.affinis (5.6g 100 g-1). Chakrabarti (2002) reported protein content of brown shrimp
244
(Metapenaues monocerous) shell waste as 8-10g 100g-1 and that in shell waste of Metapenaeus
245
endeavor was found to be 14.2 -15.2 g 100g-1 (Ariyani and Buckle, 1991). Similarly, protein
246
content in the by-products of northern pink shrimp (Pandalus borealis) was 9.3g 100g-1 and
247
that of spotted shrimp (Trachypene curvirostris) was 11.6 g 100 g-1 (Heu et al., 2003). Babu et
248
al. (2008) examined carotenoprotein contents of head waste from Penaeus monodon, M.
249
monocerus and P. indicus as 11.2 g 100 g-1, 11.3 g 100 g-1 and 12.3 g 100g-1 protein content
250
respectively.
251
3.2 Carotenoid content of proteins extracted from different shell waste
252
Carotenoid content in the shell waste from different species of shrimp varied significantly
253
(p<0.05) which is shown in Table-1. The highest carotenoid content was recorded from
254
P.stylifera shell waste (114 ± 0.02 µg g-1) while the lowest was recorded in N.tennupes (40.3 ±
255
0.01 µg g-1). In M.affinis, it was 100.6 ± 0.02 µg g-1. The carotenoid content of P.monodon
256
shell waste was 51 ± 0.02 µg g-1. Carotenoid content in crustaceans vary according to species
257
(Lambertsen et al., 1971). The carotenoid content in tiger prawns (P. monodon) from waters of
258
the Indo-Pacific region varied from 23 to 331 µg g-1 in the exoskeleton. Sachindra et al. (2005)
259
reported the carotenoid concentration in the carapace of P. monodon, P. indicus, Metapenaeus
260
dobsonii and Parapenaeopsis stylifera as 86.6, 59.8, 83.3, 104.7 µg g-1 respectively. Among
261
these, carotenoid concentration was found to be highest in the meat, head as well as carapace of
262
P. stylifera. Similar results have been obtained in the present study wherein, the carotenoid
263
content of four different shrimp shell wastes from P. monodon, M. affinis, P. stylifera and N.
264
tenuipes were 51, 100.6, 114, 40.3 µg g-1 respectively. Highest carotenoid yield was obtained
265
from the P. stylifera shell waste.
266
3.3. Amino acids profile of the carotenoprotein
267
Amino acid composition of carotenoprotein extracted from four different shell wastes are
268
represented in Table-2. Total amino acid content was highest in the carotenoprotein of P.
269
stylifera (478.30 mg g-1) and lowest was found in M.affinis (169.24 mg g-1). Amount of total
270
essential amino acid was found to be highest in P. stylifera (198.38 mg g-1). Among all amino
271
acids, glutamic acid is mainly found in higher quantity in all the carotenoproteins. Glutamic
272
acid, hydroxyproline, leucine, isoleucine, lysine, valine mainly contributes more than 50% of
273
amino acid content. Glutamine is found only in P. stylifera shell waste at a concentration of
274
1.10 mg g-1 shell waste. According to Armenta and Guerrero-Legarreta (2009),
275
carotenoproteins extracted from fermented and non-fermented Pacific white shrimp waste were
276
rich in aspartic acid and glutamic acids as well as in leucine and lysine. Carotenoprotein
277
recovered with and without the aid of bluefish trypsin contained high amount of glutamic acid/
278
glutamine (13.00 and 13.25%) and aspartic acid/ asparagines (11.18 and10.43%) (Klomklao et
279
al, 2009). Simpson and Haard (1985) also found that glutamic acid and aspartic acid were the
280
dominant amino acids in carotenoproteins isolated from shrimp wastes with and without the aid
281
of bovine trypsin. The present study reported higher carotenoprotein in SSW of P.stylifera
282
(478.3 mg g-1 shell waste) out of which, 198.38 mg g-1 was contributed by essential amino
283
acids. On the contrary, lowest amino acid content was recorded in carotenoprotein from M.
284
affinis shell waste in the present study. Similarly, Senphan et al. (2014) also reported highest
285
contribution of glutamic acid, aspartic acid, alanine, leucine, lysine, isoleucine, valine and
286
lowest content of cysteine and hydroxyproline in Pacific white shrimp (L. vannamei).
287
3.3. Antioxidant activity of different shell waste
288
DPPH ((2, 2-Diphenyl 1-picryl hydrazyl) radical scavenging activity of carotenoprotein from
289
different shell wastes are shown in Table-1. DPPH radical scavenging activity has been widely
290
used for the determination of primary antioxidant activity (Limsuwanmanee et al., 2014). The
291
present study reported highest activity in carotenoprotein extracted from P. stylifera (72.96%)
292
and the lowest in N. tenuipes (16.06%). In spite of that, carotenoids extracted from different
293
shell waste showed lower radical scavenging activity than BHA (91.4±0.008%), which is
294
normally considered as a standard for DPPH radical scavenging activity. Sila et al. (2014)
295
reported that carotenoprotein extracted from shrimp P. longirostris by-products showed higher
296
DPPH radical scavenging activity. Further, they concluded that the antioxidant activity of
297
carotenoprotein is mainly dependant on carotenoid content. Therefore, higher carotenoid
298
content in P. stylifera SSW than the other shell wastes can be correlated with better DPPH
299
radical scavenging activity in the former.
300
Ferric Reducing Antioxidant Power Assay of carotenoprotein from various shell wastes
301
is shown in Table-1 and expressed as µM Fe2+ g-1. Significant differences (P < 0.05) were
302
evident between the carotenoprotein content of different shell wastes and FRAP activity was
303
found to be highest in P. stylifera (0.979 ± 0.01 µM Fe2+ g-1), followed by M. affinis shell waste
304
(0.95 ± 0.004 µM Fe2+ g-1) while the lowest activity was obtained in P. monodon shell waste
305
(0.635 ± 0.001 µM Fe2+ g-1). Sowmya and Sachindra (2012) estimated antioxidant activity of
306
shrimp processing discards and suggested that carotenoids have a major role in the scavenging
307
of free radicals. Similar trends have also been reported in FRAP activity in the present
308
experiment, wherein highest and lowest FRAP activity was recorded in the waste from P.
309
stylifera (0.979 µM Fe2+ g-1) and N. tenuipes, respectively.
310
The ABTS radical scavenging activity assay can be applied to both lipophilic and
311
hydrophilic compounds and has been widely used as an antioxidant activity assay (Re et al.,
312
1999). 2, 2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt radical
313
scavenging activity of carotenoprotein from different shrimp shell wastes is shown in Table-1.
314
ABTS activity was found to be significantly different in all samples and varied from 163.25 ±
315
2.02 mM Trolox g-1 to 396 ± 3.92 mM Trolox g-1 among the shell wastes. It was highest in P.
316
stylifera shell waste (396.583 ± 3.92 mM Trolox g-1) and the lowest value was recorded in N.
317
tenuipes shell waste (163.25 ± 2.02 mM Trolox g-1). Higher ABTS activity in P. stylifera can
318
be explained by the occurrence of higher amount of carotenoid content in it, which helped in
319
scavenging the metal ions (Sowmya and Sachindra, 2012).
320
The above results suggest that antioxidative activities of carotenoids in carotenoproteins
321
of different shell wastes followed a concentration dependent pattern wherein P. stylifera (with
322
higher carotenoid concentration) showed higher antioxidant activity compared to all others;
323
lowest being in N. tenuipes. Carotenoid (mainly astaxanthin), has the ability to quench, trap as
324
well as scavenge free radicals and it also neutralizes free radicals by adding them into its own
325
double bond (Higuera-Ciapara, Felix-Valenzuela and Goycoolea, 2006). Thus, carotenoprotein
326
had higher radical scavenging activity as well as chelating capability when the carotenoid
327
content was higher in them, in accordance to results obtained by Sowmya and Sachindra (2012)
328
and Senphan et al. (2014). Moreover, carotenoprotein contains both carotenoid as well as
329
protein where the carotenoids and peptides in the protein act as an antioxidant. Cephalothorax
330
extracts are found to be responsible for the radical scavenging properties due to presence of
331
peptides in them (Binsan et al., 2008). Similarly, peptides from protein hydrolysates obtained
332
from shrimp shell wastes played a significant role as antioxidants (He et al., 2006).
333
Reducing power assay is mainly used to evaluate the ability of antioxidant to
334
transfer electron or hydrogen (Yildirim et al., 2003). Again, reducing power assay has
335
been found to be higher in the carotenoprotein from P. stylifera shell waste and lowest
336
in that of N. tenuipes shell waste, wherein, the OD value increased from 1.0025 to
337
3.0445, while N. tenuipes exhibited lesser absorbance from 0.866 to 1.1245 which has
338
been shown in Figure 1. Reducing power is mainly affected by bioactive compounds
339
present in shrimp shell waste, such as carotenoids, phenolic compounds,
340
chitooligosaccharides etc, which can donate electrons easily and terminate the radical
341
chain reactions (Ghorbel-Bellaaj et al., 2012). Therefore, bioactive compounds present
342
in the carotenoid and protein content of shell waste help in reducing or scavenging free
343
radicals into more stable forms thereby acting as an antioxidant. Seymour et al. (1996)
344
also reported the presence of natural antioxidants like phenolic compounds in different
345
shrimp wastes.
346
3.4 Characterization of carotenoid profile by HPLC-DAD-ESI-QTOF MS analysis
347
The carotenoprotein extracted from Parapeneopsis stylifera was evaluated for the
348
identification of carotenoid compounds by HPLC-DAD-ESI-QTOF MS analysis and the
349
HPLC chromatogram profile was shown in Figure 2. A total of 5 carotenoid derivatives,
350
including all keto carotenoid, xanthophyll and hydroxylated carotenoid were identified. Peak
351
identification was carried out based on retention time behavior and absorption spectra which
352
were shown in Table-3. All peaks in the chromatogram were detected in the retention time from
353
19.716 to 21.229 min. Peak 1 with a molecular ion at m/z 597.3905 and four fragments at m/z
354
597.3905, 598.3943, 599.398, 600.4022 corresponding to MH+ ions respectively was identified
355
as astaxanthin. Lin et al. (2005) also reported the presence of astaxanthin in spear shrimp shells
356
(Parapenaeopsis hardwickii). Three compounds (Beta-carotene, cryptoxanthin and Lutein) at
357
m/z 537.4465 and 553.4374 were categorized as xanthophyll, a derivative of carotenoid.
358
MS/MS spectra yielded three fragments corresponding to (M+H)+ and (M+Na)+ ions detected
359
in Peak 2, 4 and 5. Breithaupt (2004) also characterized the shrimp (Pandalus borealis) and
360
revealed the incidence of xanthophyll in shrimp extract. Peak 3 has retention time of 21.112
361
min, with a molecular ion at m/z 537.4465 and MS/MS spectra provided three fragments
362
resulting (M+H) + ions described as Lycopene.
363
Hence the properties of carotenoproteins described in the present work can
364
maximize the utilization of shrimp shell to cope up with the increased shell waste generation
365
which can reduce the heavy load of disposal into the ocean or landfill (Yan and Chen, 2015).
366
4. Conclusion
367
Carotenoprotein, a stable form of carotenoid, can be efficiently extracted through enzymatic
368
hydrolysis using papain enzyme, can serve as an effective antioxidant as well as being a rich
369
source of essential amino acid and carotenoid. These extracted protein-carotenoid complexes
370
can be used as nutraceuticals in feed to produce cost effective and antioxidant rich diets for
371
enhancing immunity and survival of the cultured species. Superiority of the carotenoprotein
372
extracted from shell waste of parapeneopsis stylifera has been proven in the present study in
373
terms of higher quantity of essential amino acids, carotenoids and protein content and enhanced
374
antioxidant activities as compared to the carotenoprotein extracted from shell waste of Penaeus
375
monodon, Metapenaeus affinis, Nematopalemon tenuipes. Further, research may be conducted
376
on the concept of using carotenoprotein extracted from parapeneopsis stylifera as a feed
377
ingredient, being a cost-effective nutraceutical and colour enhancer (both act as an antioxidant
378
as well as a source of carotenoids and essential amino acid).
379
Acknowledgements
380
The authors are thankful to the Director and Vice-chancellor, ICAR-Central Institute of
381
Fisheries Education, Mumbai, India for the necessary support and encouragement.
382
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Table 1. Characterization of carotenoprotein extracted from shell waste of different shrimp species (on wet weight basis) Shell waste of different shrimp species Parameter P.monodon
M.affinis
P.stylifera
N.tenuipes
Protein content (g 100 g-1)
9.80 ± 0.02d
5.60 ± 0.01a
9.20 ± 0.03c
8.90 ± 0.02b
carotenoid content (µg/ g sample)
51 ± 0.02b
114 ± 0.03d
40.30 ± 0.01a
DPPH Activity (%) FRAP activity (µM Fe2+/g) ABTS Activity (mM Trolox/g)
100.6 ± 0.02c
25.13 ± 0.01b
27.47 ± 0.01b
72.96 ± 0.04c
16.06 ±0.02a
0.63± 0.01a
0.95 ± 0.04c
0.97 ± 0.01c
0.82 ± 0.01b
294.08 ± 1.39c
200.77 ± 1.46b
396.58 ± 3.92d
163.25 ± 2.02a
Values are expressed in terms of Mean ± SE a,b,c,d values in a column with different superscripts differ significantly (p < 0.05)
Table 2. Amino acid composition of carotenoprotein from different shell wastes
Amino acid
Pm
Ps
Ma
Na
Arginine*
10.54
12.97
7.05
9.46
Threonine*
11.07
19.81
4.31
11.53
Valine*
19.14
31.41
23.79
22.08
Isoleucine *
14.99
25.70
0.00
14.85
Leucine*
24.10
39.20
26.24
24.59
Lysine*
26.70
39.72
8.18
26.60
Methionine*
4.54
8.85
8.75
4.65
Phenaylalanine*
12.31
20.72
6.37
10.62
Alanine
26.82
41.46
9.05
26.09
Aspartic acid
32.38
54.42
9.53
33.17
Glutamine
0.00
1.10
0.00
0.00
Glutamic acid
57.20
87.85
5.16
54.19
Glycine
21.23
35.68
4.61
18.71
Hydroxyproline
31.82
46.77
5.81
32.90
Serine
(mg/g shell waste)
10.06
12.64
8.10
7.86
A
123.40
198.38
84.69
124.37
TNEEB
179.51
279.93
42.25
172.92
Total Amino Acid
302.90
478.30
126.93
297.28
TEAA
*Essential amino acid in adults A
Total Essential Amino Acid
B
Total Non-Essential Amino Acid
Pm = Penaeus monodon; Ma = Metapenaeus affinis; Ps = Parapeneopsis stylifera; Nt = Nematopalemon tenuipes
Table 3. Characterization of carotenoid profile by HPLC-DAD-ESI-QTOF MS analysis
Peaks
RT (Retention Time)
Molecular formula
Structure
Mass spectrometry (m/z)
MS/MS fragments
Compound label
Major types (representative components) of carotenoid compounds
Astaxanthin
Keto carotenoid
1
19.716
C40H52O4
597.3905
597.3905 598.3943 599.398 600.4022
2
21.112
C40H56
537.4465
537.4465 538.4477 539.4437
Beta-carotene
Hydroxylated carotenoid (xanthophyll)
Lycopene
Carotenoid
3
21.112
C40H56
537.4465
537.4465 538.4477 539.4437
4
21.396
C40H56O
553.4374
553.4374 554.4483 575.4077
Cryptoxanthin
Hydroxylated carotenoid (xanthophyll)
569.4349
569.4349 570.4378 591.4085 592.4228
Lutein
Xanthophyll
5
21.229
C40H56O2
3.5
3
OD value
2.5
2
P.monodon M.affinis
1.5
P.stylifera N.tenuipes
1 0.5
0 0.5
1
2
3
4
5
concentration of carotenoprotein
Fig.1. Reducing power activity of carotenoprotein of the shell of different shrimp species *Values in bars with different superscripts differ significantly (P < 0.05)
Fig.2. Carotenoid profile of the lipid extract from P. Stylifera shrimp waste as determined by HPLC-DAD
Highlights •
Four different shrimp species shell waste were comparatively evaluated.
•
Carotenoprotein was extracted through enzymatic hydrolysis using papain enzyme.
•
Amino acid, carotenoid, carotenoid profile and antioxidant properties of four different shrimp shell waste were studied for the first time.
•
Superiority of carotenoprotein extracted from Parapeneopsis stylifera has been found.
Document type: Declaration of conflict of interest
I am herewith declaring that there is no conflict of interest in publishing the manuscript. This manuscript, or its contents in some other form, has not been published previously by any of the authors and is not under consideration for publication in another journal. All the co-authors have agreed for submission of this manuscript to your esteemed journal “Aquaculture”. Best regards